A crotoxin-specific, monoclonal, high-affinity, single-chain antibody variable region (scFv) was generated by combinatorial methods using Pharmacia's Recombinant Phage Antibody System. A high-affinity clone, designated A10G, was selected, and its DNA sequence was determined. Protein A10G showed high reaction specificity, with only the closely related rattlesnake neurotoxins, concolor toxin and Mojave toxin, showing cross-reactivity out of eleven group II phospholipase A2s (PLA2s) screened. No group I PLA2s cross-reacted in enzyme-linked immunosorbent assays. The gene coding for A10G was subcloned into an expression vector, and the resulting expressed nonfusion protein, designated A10GPE, was renatured and purified to apparent homogeneity. Dissociation constants of A10G with intact crotoxin and crotoxin basic subunit were determined to be 7 x 10(-10) and 6.8 x 10(-9) M, respectively. When A10GPE was preincubated with either the basic subunit or intact crotoxin at molar ratios of up to 5:1, no inhibition of phospholipase activity was observed. Expressed protein, however, could partially neutralize the lethality of Mojave toxin, a crotoxin homolog, in mice.